High fat diet-induced obesity prolongs critical stages of the spermatogenic cycle in a Ldlr-/-.Leiden mouse model.

Obesity can disturb spermatogenesis and subsequently affect male fertility and reproduction. In our study, we aim to elucidate at which cellular level of adult spermatogenesis the detrimental effects of obesity manifest. We induced high fat diet (HFD) obesity in low-density lipoprotein receptor knock-out Leiden (Ldlr-/-.Leiden) mice, and studied the morphological structure of the testes and histologically examined the proportion of Sertoli cells, spermatocytes and spermatids in the seminiferous tubules. We examined sperm DNA damage and chromatin condensation and measured plasma levels of leptin, testosterone, cholesterol and triglycerides. HFD-induced obesity caused high plasma leptin and abnormal testosterone levels and induced an aberrant intra-tubular organisation (ITO) which is associated with an altered spermatids/spermatocytes ratio (2:1 instead of 3:1). Mice fed a HFD had a higher level of tubules in stages VII + VIII in the spermatogenic cycle. The stages VII + VII indicate crucial processes in spermatogenic development like initiation of meiosis, initiation of spermatid elongation, and release of fully matured spermatids. In conclusion, HFD-induced obese Ldlr-/-.Leiden mice develop an aberrant ITO and alterations in the spermatogenic cycle in crucial stages (stages VII and VII). Thereby, our findings stress the importance of lifestyle guidelines in infertility treatments.

Effects of early-life stress on peripheral and central mitochondria in male mice across ages.

Exposure to early-life stress (ES) increases the vulnerability to develop metabolic diseases as well as cognitive dysfunction, but the specific biological underpinning of the ES-induced programming is unknown. Metabolic and cognitive disorders are often comorbid, suggesting possible converging underlying pathways. Mitochondrial dysfunction is implicated in both metabolic diseases and cognitive dysfunction and chronic stress impairs mitochondrial functioning. However, if and how mitochondria are impacted by ES and whether they are implicated in the ES-induced programming remains to be determined. ES was applied by providing mice with limited nesting and bedding material from postnatal day (P)2-P9, and metabolic parameters, cognitive functions and multiple aspects of mitochondria biology (i.e. mitochondrial electron transport chain (ETC) complex activity, mitochondrial DNA copy number, expression of genes relevant for mitochondrial function, and the antioxidant capacity) were studied in muscle, hypothalamus and hippocampus at P9 and late adulthood (10-12 months of age). We show that ES altered bodyweight (gain), adiposity and glucose levels at P9, but not in late adulthood. At this age, however, ES exposure led to cognitive impairments. ES affected peripheral and central mitochondria in an age-dependent manner. At P9, both muscle and hypothalamic ETC activity were affected by ES, while in hippocampus, ES altered the expression of genes involved in fission and antioxidant defence. In adulthood, alterations in ETC complex activity were observed in the hypothalamus specifically, whereas in muscle and hippocampus ES affected the expression of genes involved in mitophagy and fission, respectively. Our study demonstrates that ES affects peripheral and central mitochondria biology throughout life, thereby uncovering a converging mechanism that might contribute to the ES-induced vulnerability for both metabolic diseases and cognitive dysfunction, which could serve as a novel target for intervention.

White matter changes and gait decline in cerebral small vessel disease.

The relation between progression of cerebral small vessel disease (SVD) and gait decline is uncertain, and diffusion tensor imaging (DTI) studies on gait decline are lacking. We therefore investigated the longitudinal associations between (micro) structural brain changes and gait decline in SVD using DTI. 275 participants were included from the Radboud University Nijmegen Diffusion tensor and Magnetic resonance imaging Cohort (RUN DMC), a prospective cohort of participants with cerebral small vessel disease aged 50-85 years. Gait (using GAITRite) and magnetic resonance imaging measures were assessed during baseline (2006-2007) and follow-up (2011 - 2012). Linear regression analysis was used to investigate the association between changes in conventional magnetic resonance and diffusion tensor imaging measures and gait decline. Tract-based spatial statistics analysis was used to investigate region-specific associations between changes in white matter integrity and gait decline. 56.2% were male, mean age was 62.9 years (SD8.2), mean follow-up duration was 5.4 years (SD0.2) and mean gait speed decline was 0.2 m/s (SD0.2). Stride length decline was associated with white matter atrophy (ß = 0.16, p = 0.007), and increase in mean white matter radial diffusivity and mean diffusivity, and decrease in mean fractional anisotropy (respectively, ß = - 0.14, p = 0.009; ß = - 0.12, p = 0.018; ß = 0.10, p = 0.049), independent of age, sex, height, follow-up duration and baseline stride length. Tract-based spatial statistics analysis showed significant associations between stride length decline and fractional anisotropy decrease and mean diffusivity increase (primarily explained by radial diffusivity increase) in multiple white matter tracts, with the strongest associations found in the corpus callosum and corona radiata, independent of traditional small vessel disease markers. White matter atrophy and loss of white matter integrity are associated with gait decline in older adults with small vessel disease after 5 years of follow-up. These findings suggest that progression of SVD might play an important role in gait decline.

Butyrate restores HFD-induced adaptations in brain function and metabolism in mid-adult obese mice.

OBJECTIVE: Midlife obesity affects cognition and increases risk of developing dementia. Recent data suggest that intake of the short chain fatty acid butyrate could improve memory function, and may protect against diet-induced obesity by reducing body weight and adiposity. SUBJECTS: We examined the impact of a high-fat diet (HFD) followed by intervention with 5% (w/w) dietary butyrate, on metabolism, microbiota, brain function and structure in the low-density-lipoprotein receptor knockout (LDLr-/-) mouse model in mid and late life. RESULTS: In mid-adult mice, 15 weeks of HFD-induced adiposity, liver fibrosis and neuroinflammation, increased systolic blood pressure and decreased cerebral blood flow, functional connectivity assessed with neuroimaging. The subsequent 2 months butyrate intervention restored these detrimental effects to chow-fed control levels. Both HFD and butyrate intervention decreased variance in fecal microbiota composition. In late-adult mice, HFD showed similar detrimental effects and decreased cerebral white and gray matter integrity, whereas butyrate intervention attenuated only metabolic parameters. CONCLUSION: HFD induces detrimental effects in mid- and late-adult mice, which can be attenuated by butyrate intervention. These findings are consistent with reported associations between midlife obesity and cognitive impairment and dementia in humans. We suggest that butyrate may have potential in prevention and treatment of midlife obesity.

Sex Differences in Presynaptic Density and Neurogenesis in Middle-Aged ApoE4 and ApoE Knockout Mice.

Atherosclerosis and apolipoprotein E ε4 (APOE4) genotype are risk factors for Alzheimer's disease (AD) and cardiovascular disease (CVD). Sex differences exist in prevalence and manifestation of both diseases. We investigated sex differences respective to aging, focusing on cognitive parameters in apoE4 and apoE knockout (ko) mouse models of AD and CVD. Presynaptic density and neurogenesis were investigated immunohistochemically in male and female apoE4, apoE ko, and wild-type mice. Middle-aged female apoE4 mice showed decreased presynaptic density in the inner molecular layer of the dentate gyrus of the hippocampus. Middle-aged female apoE ko mice showed a trend towards increased neurogenesis in the hippocampus compared with wild-type mice. No differences in these parameters could be observed in middle-aged male mice. Specific harmful interactions between apoE4 and estrogen could be responsible for decreased presynaptic density in female apoE4 mice. The trend of increased neurogenesis found in female apoE ko mice supports previous studies suggesting that temporarily increased amount of synaptic contacts and/or neurogenesis is a compensatory mechanism for synaptic failure. To our knowledge, no other studies investigating presynaptic density in aging female apoE4 or apoE ko mice are available. Sex-specific differences between APOE genotypes could account for some sex differences in AD and CVD.

Limited expression of heparan sulphate proteoglycans associated with Aß deposits in the APPswe/PS1dE9 mouse model for Alzheimer's disease.

AIMS: Alzheimer's disease (AD) is characterized by deposition of the amyloid beta (Aß) peptide in brain parenchyma and vasculature. Several proteins co-deposit with Aß, including heparan sulphate proteoglycans (HSPG). HSPG have been suggested to contribute to Aß aggregation and deposition, and may influence plaque formation and persistence by stimulating Aß fibrillization and by protecting Aß against degradation. Mouse models for AD, expressing the human amyloid precursor protein (APP), produce Aß deposits similar to humans. These models may be used to study disease pathology and to develop new therapeutic interventions. We aimed to investigate whether co-deposition of HSPG in AD brains can be replicated in the APPswe/PS1dE9 mouse model for AD and if a temporal association of HSPG with Aß exists. METHODS: We studied the co-deposition of several HSPG and of the glycosaminoglycan side chains of HSPG in the APPswe/PS1dE9 model at different ages by immunohistochemistry. RESULTS: We found that, although APPswe/PS1dE9 mice did develop severe Aß pathology with age, co-deposition of HS glycosaminoglycan chains and the various HSPG (agrin, perlecan and glypican-1) was scarce (<10-30% of the Aß deposits were stained). CONCLUSIONS: Our data suggest that the molecular composition of Aß deposits in the APPswe/PS1dE9 mouse, with respect to the several HSPG investigated in this study, does not accurately reflect the human situation. The near absence of HSPG in Aß deposits in this transgenic mouse model may, in turn, hinder the translation of preclinical intervention studies from mice to men.

DHA and cholesterol containing diets influence Alzheimer-like pathology, cognition and cerebral vasculature in APPswe/PS1dE9 mice.

Cholesterol and docosahexenoic acid (DHA) may affect degenerative processes in Alzheimer's Disease (AD) by influencing Abeta metabolism indirectly via the vasculature. We investigated whether DHA-enriched diets or cholesterol-containing Typical Western Diets (TWD) alter behavior and cognition, cerebral hemodynamics (relative cerebral blood volume (rCBV)) and Abeta deposition in 8- and 15-month-old APP(swe)/PS1(dE9) mice. In addition we investigated whether changes in rCBV precede changes in Abeta deposition or vice versa. Mice were fed regular rodent chow, a TWD-, or a DHA-containing diet. Behavior, learning and memory were investigated, and rCBV was measured using contrast-enhanced MRI. The Abeta load was visualized immunohistochemically. We demonstrate that DHA altered rCBV in 8-month-old APP/PS1 and wild type mice[AU1]. In 15-month-old APP/PS1 mice DHA supplementation improved spatial memory, decreased Abeta deposition and slightly increased rCBV, indicating that a DHA-enriched diet can diminish AD-like pathology. In contrast, TWD diets decreased rCBV in 15-month-old mice. The present data indicate that long-term dietary interventions change AD-like pathology in APP/PS1 mice. Additionally, effects of the tested diets on vascular parameters were observed before effects on Abeta load were noted. These data underline the importance of vascular factors in the APP/PS1 mouse model of AD pathology.

Changes in cerebral blood volume and amyloid pathology in aged Alzheimer APP/PS1 mice on a docosahexaenoic acid (DHA) diet or cholesterol enriched Typical Western Diet (TWD).

High dietary cholesterol and low dietary docosahexaenoic acid (DHA) intake are risk factors for Alzheimer's disease (AD). However, it is unclear how these components influence the course of the disease. We investigated the effects of dietary lipids on beta-amyloid deposition and blood circulation in the brains of 18-month-old APP/PS1 mice. Starting at 6 months of age, mice were fed a regular rodent chow, a Typical Western Diet (TWD) containing 1% cholesterol, or a diet with a high (0.5%) level of DHA for 12 months. Relative cerebral blood volume (rCBV) and flow (CBF) were determined with (2)H MR spectroscopy and gradient echo contrast enhanced MRI. Deposition of beta-amyloid was visualized in fixed brain tissue with immunohistochemistry. The TWD diet increased plaque burden in the dentate gyrus of the hippocampus, but did not significantly reduce rCBV. In contrast, the DHA-enriched diet increased rCBV without changing blood flow indicating a larger circulation in the brain probably due to vasodilatation and decreased the amount of vascular beta-amyloid deposition. Together, our results indicate that the long-term intake of dietary lipids can impact both brain circulation and beta-amyloid deposition, and support the involvement of hemodynamic changes in the development of AD.

Combined uridine and choline administration improves cognitive deficits in spontaneously hypertensive rats.

Rationale. Hypertension is considered a risk factor for the development of cognitive disorders, because of its negative effects on cerebral vasculature and blood flow. Genetically induced hypertension in rats has been associated with a range of cognitive impairments. Therefore, spontaneously hypertensive rats (SHR) can potentially be used as a model for cognitive deficits in human subjects. Consecutively, it can be determined whether certain food components can improve cognition in these rats. Objective. The present study aimed to determine whether SHR display specific deficits in attention, learning, and memory function. Additionally, effects of chronic uridine and choline administration were studied. Methods. 5-7 months old SHR were compared with normotensive Wistar-Kyoto (WKY) and Sprague-Dawley (SD) rats. (a) The operant delayed non-matching-to-position (DNMTP) test was used to study short-term memory function. (b) The five-choice serial reaction time (5-CSRT) task was used to assess selective visual attention processes. (c) Finally, the Morris water maze (MWM) acquisition was used as a measure for spatial learning and mnemonic capabilities. Results. (1) SHR exhibited significantly impaired performance in the 5-CSRT test in comparison with the two other rat strains. Both the SHR and WKY showed deficits in spatial learning when compared with the SD rats. (2) Uridine and choline supplementation normalized performance of SHR in the 5-CSRT test. (3) In addition, uridine and choline treatment improved MWM acquisition in both WKY and SHR rats. Conclusion. The present results show that the SHR have a deficiency in visual selective attention and spatial learning. Therefore, the SHR may provide an interesting model in the screening of substances with therapeutic potential for treatment of cognitive disorders. A combination of uridine and choline administration improved selective attention and spatial learning in SHR.

Markers of inflammation and cellular adhesion molecules in relation to insulin resistance in nondiabetic elderly: the Rotterdam study.

Insulin resistance, which is highly prevalent in the elderly, is suggested to be accompanied by an increased acute phase response. Until now, it is unclear whether cellular adhesion molecules are involved in the clustering of insulin resistance. In the present study, we examined the relationship of insulin resistance (measured by postload insulin) with levels of markers of inflammation and cellular adhesion molecules in a random sample of 574 nondiabetic elderly men and women participating in the Rotterdam Study. Associations were assessed by regression analysis, with ln-insulin as the dependent variable [regression coefficient (95% confidence interval)]. In our population, insulin was strongly and significantly (P < 0.001) associated with the markers of inflammation C-reactive protein [1.52 (0.96-2.08)], alpha-1-antichymotrypsin [1.25 (0.82-1.69)], and IL-6 [2.60 (1.69-3.52)], adjusted for age and gender. Associations weakened, to some extent, after additional adjustment for measures of obesity, smoking, and cardiovascular disease. Insulin was associated with the soluble intercellular adhesion molecule 1 [2.22 (1.29-3.16; P < 0.001)], whereas no association with the soluble vascular cell adhesion molecule 1 was found. The strength of the associations of insulin with C-reactive protein, alpha-1-antichymotrypsin, IL-6, and soluble intercellular adhesion molecule 1, as assessed by standardized regression coefficients, was comparable with the strength of the associations of insulin with high-density lipoprotein cholesterol, body mass index, and waist-to-hip ratio. The results of this population-based study indicate that low-grade inflammation and the cellular adhesion molecule soluble intercellular adhesion molecule 1 are an integral part of insulin resistance in nondiabetic elderly. These factors may contribute to the well-known relationship between insulin resistance and cardiovascular disease risk and might potentially become therapeutic targets in insulin resistant subjects.

Subchronic mild noise stress increases HRP permeability in rat small intestine in vitro.

Recently we reported an increased trans- and paracellular protein permeability in rat small intestine after acute cold restraint stress. In the present study, we applied randomized 95- or 105-dB white noise pulses during 45 min/h, 12 h/day, duration 8 days, as a milder, but more chronic stressor to male rats. At 8 days before the noise experiments, 50% of the animals were cannulated in the vena cava for blood sampling during the experimental period. The other 50% of the animals were sacrificed at Day 9, segments of ileum were mounted in Ussing chambers and perfused at 37 degrees C. Horseradish peroxidase (HRP) was added mucosally, serosal appearance was detected enzymatically and tissues were fixed for electron microscopy. In the animals exposed to 95-dB noise, plasma corticosterone levels were enhanced twofold compared to controls, and ileal HRP flux was enhanced twofold. Electron micrographs of tissue from stressed or control animals showed no detectable paracellular staining of HRP. Quantification of HRP-containing endosomes in enterocytes revealed a twofold increase in endosome number in the animals exposed to 95-db noise indicating that the increased HRP permeability was primarily due to increased endocytosis. In contrast to the animals exposed to 95-dB noise, rats exposed to 105-dB noise showed no increase in corticosterone levels and ileal HRP fluxes were not significantly different from controls. We conclude that mild subchronic noise stress may cause a decrease in intestinal barrier function by increased transcytosis of luminal antigens.

Stress stimulates transepithelial macromolecular uptake in rat jejunum.

Evidence suggests that stress may be a contributing factor in intestinal inflammatory disease; however, the involved mechanisms have not been elucidated. We previously reported that acute stress alters epithelial physiology of rat intestine. In this study, we documented stress-induced macromolecular transport across intestinal epithelium. After exposure of Wistar-Kyoto rats to acute restraint stress, transport of a model protein, horseradish peroxidase (HRP), was assessed in isolated segments of jejunum. The flux of intact HRP was significantly enhanced across intestine from stressed rats compared with controls. Electron microscopy revealed HRP-containing endosomes within enterocytes, goblet cells, and Paneth cells of stressed rats. The number and area of HRP endosomes within enterocytes were found to be significantly increased by stress. HRP was also visualized in paracellular spaces between adjacent epithelial cells only in intestine from stressed rats. Atropine treatment of rats prevented the stress-induced abnormalities of protein transport. Our results suggest that stress, via a mechanism that involves release of acetylcholine, causes epithelial dysfunction that includes enhanced uptake of macromolecular protein antigens. We speculate that immune reactions to such foreign proteins may initiate or exacerbate inflammation.

The influence of mast cells on pathways of transepithelial antigen transport in rat intestine.

Luminal Ag challenge of intestinal segments from sensitized rats results in a rapid (approximately 3 min) secretory response. We previously showed in horseradish peroxidase (HRP)-sensitized rats that the initial phase of transepithelial Ag transport occurred via a transcellular route and was enhanced by sensitization. However, following the hypersensitivity reaction, Ag also crossed between epithelial cells. The aim of this study was to determine the role of mast cells in the altered transepithelial Ag transport. White spotting mast cell-deficient rats and +/+ littermate controls were sensitized to HRP. After 10 to 14 days, jejunal segments were resected, mounted in Ussing chambers, and challenged with HRP on the luminal side. Electron microscopy of jejunum fixed at 2 min showed a similarly enhanced endocytic transport of HRP in sensitized +/+ and Ws/Ws rats compared with naive controls. In sensitized +/+ rats, a secretory response occurred approximately 3 min after challenge, and tissue conductance increased thereafter. Naive +/+ and sensitized Ws/Ws rats did not demonstrate a secretory response to HRP challenge, and conductance remained at baseline levels. The flux of HRP was elevated across tissue from sensitized +/+ rats but not across tissue from naive controls or sensitized Ws/Ws rats. The results indicate that sensitization enhances the initial phase of transepithelial uptake of Ag by transcytosis in a mast cell-independent manner. However, subsequent recruitment of the paracellular pathway for Ag transport in sensitized rats is dependent upon the presence of mast cells and occurs after the activation of such cells.

Rapid transepithelial antigen transport in rat jejunum: impact of sensitization and the hypersensitivity reaction.

BACKGROUND & AIMS: Intestine from sensitized rats develops a rapid secretory response to luminal antigen challenge that depends on activation of subepithelial mast cells. The aim of this study was to determine the timing and route of the transepithelial protein antigen transport. METHODS: Rats were sensitized to horseradish peroxidase (HRP). After 10-14 days, jejunal segments were resected, mounted in Ussing chambers, and challenged with HRP on the luminal side. RESULTS: Electron microscopy of tissue specimens fixed at 2 minutes (before mast cell activation) showed enhanced endocytic uptake of HRP in enterocytes of HRP-sensitized rats compared with ovalbumin-sensitized or saline-injected controls. At this time, HRP was distributed throughout epithelial cells and was already evident in the lamina propria. In contrast, HRP was restricted to the apical region of enterocytes in controls. At 30 minutes (after mast cell activation), in HRP-sensitized rats only, HRP was also located within tight junctions and the paracellular region between epithelial cells. Tissue conductance was increased in HRP-sensitized rats beginning 30 minutes after HRP addition and correlated with the overall flux of HRP across the tissue. CONCLUSIONS: The results show that specific sensitization enhances the initial uptake and transcytosis of antigen across intestinal epithelium. Subsequent to activation of mast cells, antigen transport is further enhanced by penetration through the paracellular pathway.

Exocytotic release of vasoative intestinal polypeptide and serotonin from mucosal nerve fibres and endocrine cells of the intestine of the goldfish (Carassius auratus) and the tilapia (Oreochromis mossambicus): an ultrastructural study.

In earlier studies were determined the effect, presence and ultrastructure of vasoactive intestinal polypeptide (VIP) and 5-hydroxytryptamine (5-HT)-containing nerve fibres in the tilapia and goldfish intestinal mucosa. 5-HT-labelled varicosities were found close to the epithelial cells; however, synaptic membrane specializations have never been observed. VIP-like immunoreactive nerve fibres appear to be located less frequently close to the goldfish epithelium, as in the tilapia intestine, in which the distance between the VIP- or 5-HT-labelled varicosities and the epithelial cells was also rather large (more than 2 micros). To establish a possible role of VIP and 5-HT as neurotransmitters involved in the regulation of fish intestinal epithelium both electron microscopical and immunoelectron microscopical methods were used to visualize the release of 5-HT and VIP from nerve fibres. We found exocytoses from VIP-ergic and serotonergic varicosities in the muscle layers of both fish. Directly underneath the intestinal epithelium of the goldfish, it was demonstrated that 5-HT could be released from scarce varicosities. The release of 5-HT in the tilapia intestinal mucosa could only be observed from endocrine cells.

Translocation of verotoxin-1 across T84 monolayers: mechanism of bacterial toxin penetration of epithelium.

Verotoxin-producing Escherichia coli (VTEC) are pathogenic bacteria associated with diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome. Verotoxins (VTs) elaborated by these organisms produce cytopathic effects on a restricted number of cell types, including endothelial cells lining the microvasculature of the bowel and the kidney. Because human intestinal epithelial cells lack the globotriaosylceramide receptor for VT binding, it is unclear how the toxin moves across the intestinal mucosa to the systemic circulation. The aims of this study were to determine the effects of VT-1 on intestinal epithelial cell function and to characterize VT-1 translocation across monolayers of T84 cells, an intestinal epithelial cell line. VT-1 at concentrations up to 1 microgram/ml had no effect on the barrier function of T84 monolayers as assessed by measuring transmonolayer electrical resistance (102 +/- 8% of control monolayers). In contrast, both VT-positive and VT-negative VTEC bacterial strains lowered T84 transmonolayer resistance (45 +/- 7 and 38 +/- 6% of controls, respectively). Comparable amounts of toxin moved across monolayers of T84 cells, exhibiting high-resistance values, as monolayers with VTEC-induced decreases in barrier function, suggesting a transcellular mode of transport. Translocation of VT-1 across T84 monolayers paralleled the movement of a comparably sized protein, horseradish peroxidase. Immunoelectron microscopy confirmed transcellular transport of VT-1, since the toxin was observed within endosomes and associated with specific intracellular targets, including the Golgi network and endoplasmic reticulum. These data present a mode of VT-1 uptake by toxin-insensitive cells and suggest a general mechanism by which bacterial toxins lacking specific intestinal receptors can penetrate the intestinal epithelial barrier.

Carbachol, but not forskolin, increases mucosal-to-serosal transport of intact protein in rat ileum in vitro.

The effects of the secretagogues forskolin and carbachol on protein uptake in isolated ileum of rats were studied. The mucosal-to-serosal transport of horseradish peroxidase (HRP, mol mass 40 kDa) was measured in Ussing chambers, and afterwards tissues were processed for electron microscopy. In the absence of secretagogues, the flux of enzymatically active HRP was 5 pmol.cm-2.h-1 at a mucosal concentration of 10 microM. Electron micrographs showed vesicles filled with active HRP in enterocytes but no HRP activity in intercellular spaces. Forskolin decreased HRP activity in the cells. Carbachol increased the amount of HRP-filled vesicles in enterocytes and induced HRP filling in some intercellular spaces and tight junctions in the upper parts of the villi. The transepithelial flux of intact HRP increased more than 2.5-fold. This effect was suppressed by atropine. We conclude that cholinergic activation can increase the uptake of intact protein via endocytosis and the transepithelial passage by the induction of a diffusional paracellular pathway. We speculate that the increased transport of intact protein through the intestinal barrier may influence immunologic sensitization to food allergens.

Influence of forskolin and carbachol on intestinal absorption of horseradish peroxidase in the goldfish (Carassius auratus).

The transepithelial route for mucosa-to-serosa transport of the tracer macromolecule horseradish peroxidase (HRP; MW 40 kDa) and modulation of this transport by forskolin and carbachol have been studied in vi-tro in stripped goldfish intestinal epithelium mounted in Ussing-type chambers. Uptake and transport have been investigated by measuring the HRP flux from the muco-sal to serosal sides by an enzymatic method and by visualising HRP reaction products in the mucosa with electron-microscopical techniques. Both the cholinergic agonist carbachol (which is thought to increase intracellular Ca2+ and activate protein kinase C activity) and forskolin (a direct activator of adenylylcyclase) affect the amount of enzymatically active HRP in the tissue. In control tissue, HRP product is found only within the epithelial cells, the transepithelial flux reaching a constant value of about 1.5 pmoles/cm2 per h. Carbachol increases the amount of HRP product in the cells, but has no significant effect on the HRP flux compared with control values. Forskolin decreases the amount of HRP product in the cells; however, in the presence of forskolin, the lateral intercellular spaces become filled with HRP product. HRP is found in the lamina propria and the transepithelial protein flux increases more than 2.5-fold. In the presence of forskolin plus carbachol, the results are no different from the control. It is concluded that carbachol increases the endocytotic uptake of HRP, whereas forskolin inhibits the uptake but increases the paracellular permeability for HRP in goldfish intestine.

Serotonergic neurons in the intestine of two teleosts, Carassius auratus and Oreochromis mossambicus, and the effect of serotonin on transepithelial ion-selectivity and muscle tension.

The application of an antiserum directed against rat serotonin demonstrated serotonin-immunoreactive cell bodies and varicose nerve fibres in the myenteric plexus of both goldfish and tilapia. In the circular muscle layer immunoreactive varicose nerve fibres and a few cell bodies could be detected. A fine network of varicose fibres was observed underlying the epithelial cells. Serotonin immunoreactivity was not observed along incoming mesenterial blood vessels, suggesting that the serotonergic neurons may be intrinsic to the intestine. Immunoreactive endocrine cells were shown in the intestinal epithelium of tilapia but not in goldfish. Serotonin caused a weak contraction of the intestinal wall of the goldfish which could be blocked by tetrodotoxin and by atropine, suggesting that serotonin has an indirect action on muscular contraction. In contrast, serotonin induced a relaxation of the intestinal wall of tilapia which could not be blocked by propranolol or by tetrodotoxin. This indicates that in this species serotonin may act directly on the muscle fibres. The ion-selectivity of the intestinal epithelium of both species was modulated by serotonin. Tetrodotoxin did not inhibit this effect, suggesting that serotonin acts directly on the epithelial cells. The presence of serotonergic fibres in the muscle layer and directly underneath the epithelium, along with the effect of serotonin on muscular tension and on the ion-selectivity of the epithelium, suggests that serotonin may play a role in the regulation of motility and the epithelial function of goldfish and tilapia intestine.